Hydrogen Peroxide
Hydrogen peroxide, or H2O2, is naturally found in human cells and is used for disinfection. Hydrogen peroxide has anti-fungal, anti-bacterial, anti-parasitic and anti-viral activity. Once hydrogen peroxide interacts with certain enzymes, catalase or peroxidase, it yields water and an oxygen ion. Two singlet oxygen ions make stable oxygen, O2.
The bacterial origin of dental disease is well established. Dental caries, gingivitis and halitosis are all associated with resident oral bacteria. Reduction in their number by germicidal agents is a current aim to control these oral conditions. Glucose oxidase, a flavo-enzyme (GLU-OXY), is known to act upon a glucose substrate to produce hydrogen peroxide and gluconic acid. Since the mouth also contains highly active proteases, any additional oxidase enzyme can be rapidly inactivated by proteolytic action of these protease enzymes.
Glucose oxidase (B-D-glucose: oxygen I-oxidoreductase. ECI.1.3.4.) flavoenzyme is widely used for the determination of glucose in body fluids, and in removing residual glucose and oxygen from beverages and foodstuffs. See William. D. C., or al Clin. Chem. 22, 372, 1976. Furthermore, glucose oxidase-producing molds, such as Aspergillus and Penicillium species, have been used for the biological production of gluconic acid, as well as the oxidase. A unit is defined as the enzyme amount which causes oxidation of one micromole of glucose per minute at 25 C and pH 7.0.
Glucose oxidase combined with an appropriate cofactor catalyses the oxidation of R-D-glucose to D-glucono-1, 5-lactone and hydrogen peroxide, using molecular oxygen as the electron acceptor. The pH range is from 4 to 7, with an optimum pH at 5.5. The enzyme, GLU-OXY, is a dimeric holoprotein with a molecular weight of 160 (KDA 160.000 Daltons), containing one tightly bound (Kw 1×10-10) flavin adenine dinueleotide (FAD) per monomer, as cofactor. The FAD is not covalently bound and so can be released from the holoprotein following denaturation.
The human oral cavity° is known to contain other highly active proteolytic enzymes, known as proteases, and concurrently, the afore-described glucose oxidase. The latter is useful to act upon glucose to produce gluconic acid and hydrogen peroxide. The peroxide, in turn, is known to be converted by oral catalase to oxygen and water. The liberated oxygen has a germicidal effect upon the oral cavity. If the oral GLU-OXY concentration is increased, for example, by addition to a dentifrice, the proteolytic enzymes will interfere with enhanced level of glucose oxidase enzyme, by destroying the glucose oxidase. So, there is a need to protect glucose oxidase from the ever present oral proteolytic enzymes. The same utility of Glucose Oxidase can be found in the mucosa, in open wounds and within other body cavities such as the vagina, the nasal and auditory canal, the urethra and induced opening such as venous punctures.
Proteolytic enzymes are large group of natural proteins (polypeptides) with a peptide chain coiled to form an alpha-helix, and which display enzymatic activity; that is, they catalyze specific organic, or even inorganic, reactions. A typical proteolytic protein-splitting enzyme is alpha-chymotrypsin of a molecular weight c.25,000. It has a quite general power of affecting the hydrolysis of carboxyl derivatives, amides, esters, and hydrazides.
Attempts to exploit these natural antimicrobial systems have been directed to both the oral care field and the gastrointestinal tract. U.S. Pat. No. 4,150,113 and U.S. Pat. No. 4,178,362 (Hoogendom, et al) describe dentifrice compositions containing glucose oxidase, that react with plaque and salivary glucose to produce low levels of hydrogen peroxide. Hydrogen peroxide production by such systems is however, highly irregular, due to the non-uniform distribution and unpredictable availability of substrate, namely glucose, in the oral cavity, and instability of the enzyme in the presence of proteases. The effects are described as transient due to the inherent instability of the glucose Oxidase enzyme.
U.S. Pat. No. 4,269,822. U.S. Pat. No. 4,564,519, and U.S. Pat. No. 4,578,265 (Pellico, etal) further describe dentifrice compositions containing an oxido-reductase enzyme and its specific substrate in an aqueous solution for the purpose of producing in the oral cavity hydrogen peroxide or other antimicrobial oxidizing compounds such as hypothiocyanite ion. A more predictable amount of hydrogen peroxide (and subsequently hypothiocyanite ions) is produced by the compositions of Pellico et al, compared with those of the Hoogendorn references (U.S. Pat. Nos. 4,150,113 and 4,178,362). The differences between the two compositions reflect the availability of glucose in the oral cavity as substrate for glucose oxidase.
U.S. Pat. No. 4,564,519 describes a chewable dentifrice, such as a chewing gum or lozenge, which contains a dual enzymes system for producing hypothiocyanite ions upon being chewed or otherwise activated by the moisture in saliva. Such compositions stiffer from similar drawbacks to those mentioned immediately above, namely a slow rate of enzymatically-produced hydrogen peroxidase as well as a reliance on a cariogenic compound.
The formation of calculus and dental plaque is the primary source of gingivitis, dental caries, periodontal disease, tooth staining and tooth loss.
Dental calculus, or tartar as it is sometimes called, is a deposit, which forms on the surfaces of the teeth at the gingival margin. Mature calculus consists of an inorganic portion which is largely calcium phosphate arranged in a by hydroxyapatite crystal lattice structure similar to bone, enamel and dentine. An organic portion is also present and consists of desquamated epithelial cells, leukocytes, salivary sediment, food debris and other various types, unless stained or discolored by some extraneous agent. In addition to being unsightly and undesirable from an aesthetic standpoint, the mature calculus deposits are constant sources of irritation of the gingiva,
Plaque can be defined as a complex microbial community, with bacteria comprising approximately 70-80% of the plaque matrix. It has been estimated that as many as 400 distinct bacterial species may be found in plaque. This mix includes both aerobic and anaerobic bacteria, fungi, and protozoa. Viruses have also been found in samples of dental plaque. In addition to the bacterial cells, plaque contains a small number of epithelial cells, leukocytes, and macrophages. The cells are contained within an extra cellular matrix, which is formed from bacterial products and saliva. The extra cellular matrix contains protein, polysaccharide and lipids. This matrix of organisms and oral exudates continues expanding and coalesces with other plaque growths situated nearby. The bacteria synthesize levans and glucans from sucrose found in the oral cavity providing energy for the microorganisms. These glucans, levans, and microorganisms form an adhesive skeleton for the continued proliferation of plaque.
Retarding and/or stopping the proliferation of plaque and calculus are critical to maintaining good oral health. Plaque and calculus formation may lead to dental caries, gingival inflammation, periodontal disease, and ultimately tooth loss. Additionally, calculus and plaque along with behavioral and environmental factors lead to formation of dental stains, significantly affecting the aesthetic appearance of teeth. Behavioral and environmental factors that contribute to teeth staining propensity include regular use of coffee, tea, wine, cola or tobacco products.
Plaque has been classified by association with disease state as “health-associated” or “disease associated”. The latter classification is related to differences in the microbial composition of dental plaque in health versus disease. A newly cleaned tooth surface is rapidly covered with a glycoprotein deposit referred to as “pellicle”. The pellicle is derived from salivary constituents which are selectively adsorbed onto the tooth surface. The formation of pellicle is the first step in plaque formation.
The pellicle-coated tooth surface is colonized by Gram-positive bacteria such as Streptococcus sanguts, Streptococcus mutans, and Aetinornyces viscosus. These organisms are examples of the “primary colonizers” of dental plaque. Bacterial surface molecules interact with components of the dental pellicle to enable the bacteria to attach or adhere to the pellicle-coated tooth surface. Within a short time after cleaning a tooth, these Gram-positive species may be found on the tooth surface. After the initial colonization of the tooth surface, plaque increases by two distinct mechanisms: 1) the multiplication of bacteria already attached to the tooth surface, and 2) the subsequent attachment and multiplication of new bacterial species to cells of bacteria already present in the plaque mass. These new bacteria include anaerobic Grain-negative species such as Fusobacterium nucleatum and Prevotella intetnzedia; and the Capnocytophaga species. The overall pattern observed in dental plaque development is a very characteristic shift from the early predominance of Grain-positive facultative microorganisms to the later predominance of Gram-negative anaerobic microorganisms, as the plaque mass accumulates and matures. This developmental progression is also reflected in the shifts in predominant microorganisms that are observed in the transition from health to disease. Studies of plaque taken from sites of health or disease and examined either microscopically or by culturing have demonstrated distinct differences in health versus disease-associated microbial populations.
Halitosis has also been an unsolved physiological problem for centuries, and remains as such in the modern era. Halitosis is the technical term for bad breath, a condition estimated to affect 50 to 65% of the population. Up to 90% of cases are thought to originate from sources in the mouth, including poor oral hygiene, periodontal disease, coating on the tongue, impacted food, faulty dental restoration, and throat infection. The chemical basis of halitosis lies in the concentration of mouth-bound volatile and odiferous compounds, primarily organic and inorganic sulfides as well as organic amines. These odiferous volatiles are biologically synthesized by particular microorganisms that reside in the oral cavity. Halitosis is primarily caused by certain anaerobic strains of bacteria (Rosemberg, M., Bad Breath: Research Perspectives. Rumor Publishing, 1995). Specifically, the proliferation in saliva of the anaerobic bacterial pathogen Fusobacterium Species, in combination with other anaerobes, has been shown as the major biological source of halitosis.
Factors that support the growth of these bacteria will predispose a person to halitosis. Examples include accumulation of food within pockets around the teeth, among the bumps at the back of the tongue, or in small pockets in the tonsils; sloughed cells from the mouth; and diminished saliva flow. Mucus in the throat or sinuses can also serve as a breeding ground for bacteria. Conditions are most favorable for odor production during the night and between meals.
Although bad breath primarily represents a source of embarrassment or annoyance, research has shown that the sulfur gases most responsible for halitosis (hydrogen sulfide and methyl mercaptan) are also potentially damaging to the tissues in the mouth, and can lead to periodontal disease (a bacterial infection of the gains and ligaments supporting the teeth). As periodontal disease progresses, so may the halitosis, as bacteria accumulate in the pockets that form next to the teeth.
The ultimate oral cleaning level is what dentists provide during prophylaxis; daily oral care at home requires products with multiple ingredients working by different mechanisms to provide satisfactory cleaning and whitening, Oral care products for daily use such as dentifrice and rinses provide overall cleaning, but it is necessary to add ingredients for provision of anti-plaque and anti calculus benefits as well as breath freshening, stain removal, stain control and tooth whitening. Such ingredients for removal and control of stain and for whitening include bleaches, abrasives or chemical chelants. Bleaches added to dentifrices are typically present in low concentrations due to stability and safety limits unique to toothpastes. At these low concentrations bleaches, typically oxidizing agents, are generally ineffective at tooth whitening and stain control. Dental abrasives provide whitening benefits on ‘brushed’ areas of teeth, but unfortunately are of limited effect in controlling aesthetically undesirable stains that Joint along the gumline and interproximally.
Although products containing chemical oxidizing agents and other plaque and calculus reduction agents are known, there is a continuing need to develop improved products, in particular products that provide enhanced overall cleaning by concurrently attacking the calculus, plaque, and staining problems.
Attempts to exploit these natural antimicrobial systems have been directed to both the oral care field and the gastrointestinal tract. U.S. Pat. No. 4,150,113 and U.S. Pat. No. 4,178,362 (Hoogendom, et al) describe dentifrice compositions containing glucose oxidase, that react with plaque and salivary glucose to produce low levels of hydrogen peroxide. Hydrogen peroxide production by such systems is, however, highly irregular, due to the non-uniform distribution and unpredictable availability of substrate, namely glucose in the oral cavity and instability of the enzyme in the presence of protease.
U.S. Pat. No. 4,269,822, U.S. Pat. No. 4,564,519, and U.S. Pat. No. 4,578,265 (Pellico, et at) further describe dentifrice compositions containing an oxidoreductase enzyme and its specific substrate in an aqueous solution for the purpose of producing in the oral cavity hydrogen peroxide or other antimicrobial oxidizing compounds such as hypothiocyanite ion. A more predictable amount of hydrogen peroxide (and subsequently hypothiocyanite ions) is produced by the compositions of Pellico et al, compared with those of the Hoogendom references (U.S. Pat. Nos. 4,150,113 and 4,178,362). The differences between the two compositions reflect the availability of glucose in the oral cavity as substrate for glucose oxidase.                U.S. Pat. No. 4,564,519 describes a chewable dentifrice, such as a chewing gum or lozenge, which contains a dual enzymes system for producing hypothiocyanite ions upon being chewed or otherwise activated by the moisture in saliva. Such compositions suffer from similar drawbacks to those mentioned immediately above, namely, a slow rate of enzymatically-produced hydrogen peroxidase as well as a reliance on a cariogenic compound.Background Oral Proteases        
The oral cavity contains many specific and non-specific proteases, both endogenous and exogenous. Exogenous types are from both viral and bacterial sources, while endogenous protease are mainly secreted by salivary glands. Some of these proteases include: three enzymes including leucine amino peptidase, dipeptidyl peptidase IV and trypsin-like proteinase, salivary matrix metalloproteinases (MMPs) (may participate in the pathogenesis of mucosal lesions and dentinal caries), particularly activity of MMP- (collagenase-2) and MMP-9 (gelatinase B), cysteine peptidases, aminopeptidase, neuropeptide-degrading enzymes and secreted aspartic proteases (Saps) These enzymes destroy a wide variety of proteins, including other enzymes.
Degradation of Glucose Oxidase by Salivary Proteases
Glucose oxidase in the presence of glucose and oxygen will convert glucose to hydrogen peroxide and gluconic acid at a fast rate, (GOx from Aspergillus nigec Michaelis constant KM 33 The Michaelis constant Kin is defined as the substrate concentration at ½ the maximum velocity) By measuring the oxygen consumption the reaction rate can be followed by using a Clark oxygen electrode. Three test cells were set up containing five milligrams of the enzyme in 10 ml of saline at pH 7.0. Test cell one contained only saline and enzyme, cell two contained enzyme plus five milligrams of commercial bacterial protease from Bacillus polymyra (1 unit/milligram), and cell three contained glucose oxidase enzyme plus two milliliter of human saliva. The cells were incubated qt 37 degrees Centigrade for 10 minutes. Two hundred millimoles of glucose were then added to each cell and the oxygen consumption measured over five minutes.
Results
Cell one showed 52% of the oxygen consumed in 5 minutes, cell two showed 10% of the oxygen consumed in five minutes and cell three showed 8% of the oxygen consumed in five minutes. These results confirmed the degradation of glucose oxidase by oral proteases.
Immobilized Glucose Oxidase Protection from Salivary Protease
Immobilization of enzymes is an established procedure in biochemistry, many factors affect the choice of substrate on which to fix the enzyme, it is often convenient to immobilize the enzyme on a support material by adsorption, deposition or a chemical means. Useful supports are porous glass, celite, porous hydrophobic resins and ion exchange materials. Depending on the properties of the support (particle size, pore size, etc) and other parameters, the enzyme loading is variable. In addition the solvent can affect the enzyme catalyzed reaction by influencing the solvation of the substrates and products or by direct interaction with the enzyme. Water is the main solvent in saliva.
Effects of the Water Content
Even in biocatalytic systems which contain mainly organic solvent and/or organic substrates, the catalytic activity is highly dependent on the amount of water present. The amount of water is best quantified in terms of water activity since the water activity is correlated with the hydration of the enzyme which in turn governs the catalytic activity. Salvia is 90+% water, so we can consider the solvent system as water.